Inhibition of clathrin assembly by high affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP- 3

W. Ye, N. Ali, M. E. Bembenek, S. B. Shears, E. M. Lafer

Research output: Contribution to journalArticlepeer-review

155 Scopus citations

Abstract

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (K(d) = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (K(d) = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol- 1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 μM, inositol- 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 μM, inositol-1,3,4,5- tetrakisphosphate (Ins(1,3,4,5)P4) > 10 μM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 μM. Moreover, 10 μM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.

Original languageEnglish (US)
Pages (from-to)1564-1568
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number4
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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